Embryonic stem cells (ES cells) are capable of differentiating into various types of cells even in vitro. In vitro differentiation of ES cells is performed by floating culture to form pseudo-embryos, called embryoid bodies, or by coculture with cells, such as stromal cells, that support differentiation and proliferation of ES cells. It is known that ES cells differentiate into various types of cells when the cells are cultured to high density without LIF (Leukemia Inhibitory Factor), and then floating cultured so as not to adhere to a culture vessel, such as a petri dish, to form cell aggregates. The cell aggregates formed by floating culture are called embryoid bodies (EB), and the floating culture is the most common method for differentiating ES cells in vitro.
An embryoid body has a ball-like structure composed of a bilayer of cells. The outer layer corresponds to visceral endoderm, the inner layer corresponds to embryonic ectoderm, and the two endoderms are separated by a basement membrane. This structure is quite similar to that of a cylindrical embryo, which is a day 6 mouse embryo. As far as this similarity is concerned, this structure resembles the normal stage of embryogenesis. In embryoid bodies, mesoderm is also induced, and cardiomyocytes, blood cells, and even primitive vascular networks are developed. When plated on a culture petri dish and cultured further, the embryoid bodies differentiate into various types of cells, including neurons, keratinocytes, chondrocytes, adipocites, and the like. It has recently been confirmed that the cells that differentiate via formation of embryoid bodies are differentiated not only into somatic cells, but also into a germ cell lineage. As such, formation of embryoid bodies is useful for demonstrating pluripotency of ES cells.
For embryoid formation, so-called a “hanging drop method” is widely used, which is devised to prevent adhesion of ES cells to a culture vessel. There are known hanging drop method 1, wherein ES cells are added to and cultured in the drops having from the lid of a glass container, and hanging drop method 2, wherein ES cells are placed over mineral oil previously placed in a culture vessel, and cultured. In hanging drop method 1, however, the hanging drops must be prevented from falling which causes extreme complexity in culture preparation and handling. In hanging drop method 2 using mineral oil, on the other hand, the interface between the mineral oil and the overlaid cell suspension must be prevented from being disrupted, and also no microscopic examination is allowed before the generated embryoid bodies are transferred to another culture vessel, which impedes researches in embryogenesis.
Phosphorylcholine group-containing polymers have been revealed to have properties ascribable to their phospholipid-like structure originated from biomembranes, such as blood compatibility, complement activation, and nonadsorbability of biomaterials, and development of bio-related materials making good use of such functions has been actively made. For example, Patent Publication 1 discloses a method of producing 2-methacryloyloxyethyl phosphorylcholine (abbreviated as MP/C hereinbelow) and excellent biocompatibility of polymers thereof. Patent Publication 2 discloses usefulness of copolymers of MP/C and methacrylate as medical materials due to their ability to hardly allow platelet adhesion or aggregation and plasma protein adhesion. Patent Publication 3 discloses medical materials prepared from a copolymer having a phosphorylcholine-like group in its side chain. Patent Publications 4 and 5 disclose excellent biocompatibility achieved by coating a resin surface with a polymer having a phosphorylcholine-like group. Patent Publication 6 discloses a separating agent and a method of separation and collection for separating and collecting blood cells, cell lines, or primary culture cells, using polyethylene terephthalate coated with a polymer having a phosphorylcholine-like group.
It is not known, however, to use a vessel coated with a polymer having a phosphorylcholine-like group, for floating culture of ES cells.
Patent Publication 1: JP-54-36025-A
Patent Publication 2: JP-3-39309-A
Patent Publication 3: JP-9-183819-A
Patent Publication 4: JP-6-502200-A
Patent Publication 5: JP-7-502053-A
Patent Publication 6: JP-2002-098676-A